Our goal during the past thirty years of NIH funding has been to understand the mechanism of chemotaxis in the Gram-positive bacterium Bacillus subtilis. During the past decade, the realization has emerged that the B. subtilis paradigm might be the ideal one for understanding these diverse mechanisms in broad sweep of bacteria and archaea because of the organism[unreadable]s relative complexity and inclusion of most known chemotaxis genes. The B. subtilis chemotaxis pathway is also similar to the one used in many archaea and the primitive thermophile Thermotoga maritima. Thus, in studying this mechanism, we can understand how chemotaxis in bacteria and archaea might have evolved and gain insights into how these diverse pathways may function. During the last funding period, we have made tremendous progress in elucidating the biochemical function of three proteins not found in Escherichia coli chemotaxis, CheC, CheD, and CheV. Collectively, this work has enabled us to formulate a working hypothesis for how the integrated pathway functions. The key element of this hypothesis is that there are three adaptation systems in B. subtilis (unlike just one in E. coli). In addition to the previously identified methylation and CheV systems, we have discovered a third adaptation system involving CheY, CheC, and CheD. Furthermore, we now have a molecular model for how the selective methylation of specific residues on the receptors differentially affects CheA kinase activity. These breakthroughs have been greatly facilitated by the recent structures for T. maritima CheC, CheD, and the cytoplasmic domain of TM1143 (a chemoreceptor). As a result, we are now in a position to propose detailed experiments to elucidate key mechanisms in B. subtilis chemotaxis. Specifically, we wish to address three questions: How does selective receptor methylation tune kinase activity in response to positive and negative stimuli? How do the receptors transduce signals from the exterior of the cell to the CheA kinase? How does CheD control receptor activity? To investigate methylation, we will measure the state of each methylated residue as a function of time in response to the addition and removal of attractants. We will also test our molecular model for selective methylation by measuring the behavior of different mutants and the underlying conformational changes in the receptor using disulfide crosslinking. Particular focus with be directed toward understanding the putative role of zinc in coordinating electrostatic interactions between neighboring methylation sites. To investigate transmembrane signaling, we will explore the role of receptors interactions occurring at the HAMP domain and extracellular ligand-binding domain. A key prediction of our model is that attractant causes the rearrangement of dimers within the receptor ensemble. To understand how CheD controls receptor activity as part of CheC-CheY feedback loop, we will identify the CheD binding sites on the receptors. Locating these sites will help explain how CheD is able to tune receptor activity.